A critical roadblock in the development of an HIV-1 vaccine is our current inability to deliver HIV-1 antigens efficiently to the immune system and to prime predictable, high frequency immune responses in humans. Recombinant Ad5 vector-based vaccines have elicited potent immune responses in preclinical studies. However, the high frequency of anti-Ad5 immunity in the developing world will likely limit the immunogenicity and clinical utility of rAd5 vaccines. We therefore propose the development of novel, replication-incompetent rAd vector-based vaccines for HIV-1. In this project, we will investigate the hypothesis that novel rAd vector-based vaccines derived from rare Ad serotypes and engineered for improved immunogenicity will elicit potent immune responses in rhesus monkeys with anti-Ad5 immunity. We further hypothesize that heterologous rAd prime-boost regimens utilizing these novel rAd vectors will prove substantially more immunogenic than traditional rAd5 vaccines in the presence of anti-Ad5 immunity. As a key component of these studies, we will systematically compare the immunogenicity and protective efficacy of various rAd vector combinations to determine the optimal vaccine regimen to advance into the clinical studies described in Project 2 of this IPCAVD grant. To investigate these hypotheses, we propose the following five Specific Aims: 1. To compare the immunogenicity of rAd5, rAd35, and capsid chimeric rAd5/rAd35 vectors in rhesus monkeys with anti-Ad5 immunity; 2. To compare the immunogenicity of various heterologous rAd prime-boost regimens in rhesus monkeys with anti-Ad5 immunity and to assess their protective efficacy against a SHIV-89.6P challenge; 3. To assess the immunogenicity and protective efficacy of the optimal heterologous rAd prime-boost regimen against an SIVmac251 challenge in rhesus monkeys with and without anti-Ad5 immunity; 4. To assess the immunogenicity and protective efficacy of limiting vaccine doses of the optimal heterologous rAd prime-boost regimen against a heterologous SIVmac251 challenge in rhesus monkeys with anti-Ad5 immunity; and 5. To determine the protective efficacy of the optimal heterologous rAd prime-boost regimen against repetitive, low-dose, mucosal, heterologous SIVmac251 challenges in rhesus monkeys with anti-Ad5 immunity.